Expression of TFPIb in Normal Human Tissues and Inhibitory Activity on Tissue Factor-Initiated Coagulation Reactions by Recombinant TFPIb from Transfected CHO Cells and Escherichia Coli

Abstract number: P1953

Moatti D, Andrade M, Tunstead J, Hathcock J, Nemerson Y

Tissue factor pathway inhibitor a (TFPIa), known as TFPI-1, is the major down regulator of tissue factor (TF): factor VIIa (FVIIa) procoagulant activity. TFPIb, an alternatively spliced form of TFPIa, lacks the Kunitz 3 domain and C-terminus of TFPIa, and instead contains an unrelated C-terminus. We have explored TFPIb expression in human tissues and inhibitory activity of TFPIb expressed in Escherichia coli (E. Coli) and CHO cells. Reverse transcriptase polymerase chain reaction, western blotting and immunohistochemistry demonstrated the presence of TFPIb in several human tissues including heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas. Recombinant TFPIb produced in E. Coli significantly inhibited factor Xa formation in a concentration dependant manner when incubated with lipidated TF, factors X, VIIa and Ca²⁺. Moreover, CHO cells were transfected with PtriEx-3 Neo plasmid containing the TFPIb insert and the cells that expressed TFPIb showed a significant decrease of TF activity (P < 0.005) and a significant increase of clotting time (P= 0.001) compared to untransfected CHO cells or CHO transfected with the plasmid only. Our results showed the expression of TFPIb in normal human tissues and inhibitory activity of recombinant TFPIb.